Bovine monoclonal antibodies to bovine herpesvirus I from sequential fusion heterohybridomas

ABSTRACT

Stable heterohybridomas which secrete monoclonal antibodies to bovine herpesvirus-1 (BHV-1) are obtained by fusing primed bovine lymphocytes with an immortal cell line, selecting for secretial hybrids, and sequentially re-fusing such hybrids with fresh bovine lymphocytes. Three such heterohybridomas which secrete neutralizing antibody to BHV-1 have been obtained in this manner and have been deposited in the ATCC as Accession Nos. HB 9907, HB 9908, and HB 9909. The neutralizing antibodies are useful in immunological research, pathological diagnosis, and therapeutic disease control. Specifically, they have utility as serologic control reagents in assays, and as primary reagents in anti-species, anti-isotype, and anti-idiotypic antibody production.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a novel class of bovine-murine hybridomaswhich produce neutralizing monoclonal antibodies to bovine herpesvirus-1(BHV-1). BHV-1 is the agent responsible for infectious pustularvulvovaginitis and bovine rhinotracheitis. Secondary infectionsassociated with rhinotracheitis may develop into an economicallydevastating disease known as "shipping fever." Efforts to controldiseases induced by BHV-1 have been hindered for want of reliablediagnostic reagents and effective therapeutic agents.

2. Description of the Prior Art

Attenuated vaccines which have been developed against BHV-1 aretypically characterized by undesirable side effects, and the killedvaccines are largely ineffective. Lupton et al. [Am. J. Vet. Res. 41:383 (1980)] have reported on a crude subunit vaccine for protectingcalves against the infection and shed of infectious BHV-1. Van Drunen etal. [J. Virol. 59: 401 (1980)] and Babiuk et al. [Virology 159: 57(1987)] have shown that the administration of each of three majorenvelope glycoproteins of BHV-1 to calves will protect against combinedchallenge with BHV-1 and Pasteurella haemolytica, but not against viralreplication and shedding. Passively administered, murine, monoclonalantibodies specific for epitopes on the three envelope glycoproteinswere found by Marshall et al. to be nonprotective against BHV-1infection in calves.

Presumably, monoclonal antibody of bovine origin would have advantagesin many applications, including: the determination of key epitopesresponded to by the host; cooperation between antibody and hostaccessory immunologic cells; repeated prophylactic or therapeuticadministration of antibodies to the host; production of some types ofanti-idiotypic antibodies; and as control reagents for serologic assays.For example, Srikumaran et al. [Abstract of Papers, 67th Annual Meetingof the Conference of Research Workers in Animal Disease, 86: Abstr. No.143 (1986)] fused lymph cells from a calf immunized against infectiousbovine rhinotracheitis virus (IBRV) with the nonsecreting cell lineSP2/0. Two of the recovered hybridomas secreted nonneutralizing, bovinemonoclonal antibodies that reacted with IBRV in both indirectsolid-phase radioimmunodiffusion assay (RIA), and indirect fluorescentantibody assay (IFA). Tucker et al. [Hybridoma 3(2): 171 (1984)]demonstrated that antibody secretion vigor of interspecies hybridomascould be enhanced by re-fusing a chemically selected mouse/calfhybridoma with immunized calf lymph node cells. In contrast to theoriginal mouse/calf fusion products which failed to maintain antibodysecretion in culture, the re-fused lines carried two to three times thenumber of bovine chromosomes as the single-fused hybridoma andmaintained active antibody secretion over a 5-month period.

SUMMARY OF THE INVENTION

We have now discovered that by fusing bovine lymphocytes with cells froman immortal cell line and then sequentially re-fusing the resultantheterohybridomas which have been selected for secretion of monoclonalantibody to BHV-1, stable lines can be obtained for producingneutralizing antibody to the virus. Specifically, three such lines havebeen obtained from a triple fusion sequence. In the first fusion of theseries, lymph node cells from a calf hyperimmunized with BHV-1 werefused with the nonsecretor murine hybridoma SP2/0, and first-generationmonoclonal antibody-producing, bovine x murine, heterohybridomas wereselected. These first-generation heterohybridomas were then re-fusedwith primed lymph node cells similar to those used in the first fusion,and second-generation monoclonal antibody-producing, bovine² x murine,heterohybridomas were selected. Finally, the second-generationheterohybridomas were fused with primed lymph node cells and a thirdgeneration of bovine³ x murine heterohybridomas were selected. Of theresultant triple fusion products which were found to be stable,approximately 4% were sufficiently bovine in nature so as to secreteIgG1 antibodies which specifically neutralized BHV-1 in vitro withoutcomplement.

In accordance with this discovery it is an object of this invention toprovide a method, using re-fusion technology, for obtaining stable,continuously proliferating, hybrid cell lines capable of producingneutralizing antibodies to BHV-1.

It is also an object of the invention to provide three specific bovine³x murine heterohybridomas for producing neutralizing antibodies toBHV-1.

Another object of the invention is to provide a source for bovineantibodies to BHV-1 which offer unique advantages over host polyclonalantiserum or murine monoclonal antibodies in immunological research,pathological diagnosis, and therapeutic disease control.

A specific object of the invention is to produce bovine monoclonalantibodies which have utility as serologic control reagents in assays,and as primary reagents in anti-species, anti-isotype, and especiallyanti-idiotypic antibody production.

Other objects and advantages of the invention will become readilyapparent from the ensuing description.

DETAILED DESCRIPTION OF THE INVENTION

Establishing the antibody-secreting cell lines for use in the inventionis a multistep procedure which includes hyperimmunizing a bovine animalto induce a proliferation of antibody-producing cells, promoting fusionbetween the primed bovine cells and cells of an immortal cell line,selecting for antibody-secreting heterohybridomas, screening thehybridomas for selectability in a subsequent fusion stage, and thensequentially re-fusing the selected hybridomas with fresh, primed bovinecells to enrich the bovineness of the recovered cell lines. Theobjective is to produce cell lines which are continuous (i.e., immortal)and stable. The term "stable" is used herein to describe the property ofcontinuously producing antibody.

The immortal cell line may be of any origin as known in the art but ismost desirably one which does not itself secrete antibody. Immortal celllines most commonly used in fusion technology are of murine origin,especially those which are hypoxanthine-guanine, phosphoribosyltransferase (HGPRT) negative. Particularly preferred for use herein isthe SP2/0 myeloma established from a BALB/c mouse by Shulman et al.[Nature 276: 269-270 (Nov. 16, 1978)]. An objective of the fusionprocedure is to maximize the number of myeloma cells by passaging theline on a suitable growth medium according to a schedule allowing forthe log phase to coincide with the fusion date. The cell line is alsopassaged two or three times at 4- to 8-week intervals on a growth mediumcontaining 6-thioguanine and 8-azaguanine or the like for the purpose ofmaintaining susceptibility to a screening agent during the hybridomaselection, as described in further detail below.

Bovine cells for use in the fusion are HGPRT-positive lymphocytes whichhave been primed for antibody production to BHV-1 by hyperimmunizationof an animal with BHV-1 antigen. Lymph node cells of a calf are asuitable source for these lymphocytes, and they are most optimallyprimed by injection of the BHV-1 antigen directly into the region of thenode itself. Live, low passage BHV-1 in a suitable adjuvant is aneffective antigen for this purpose. Of course, it would be understood bya person in the art that the purer the antigenic material used for thehyperimmunization, the more specific the lymphocytic response and themore facile the subsequent hybridoma selection process.

It is theorized that there is a certain advantage to using primedlymphocytes from the same individual animal for each of the sequentialre-fusions. Lymphocytes from the same animal have similarhistocompatibility antigens. This approach will hereafter be referred toas "selfing." For selfing to be implemented, the cells either have to benonsacrificially recovered from the animal prior to each fusion, or elsethey have to be recovered at one time and maintained in the primed statesuch as by cryogenic preservation or in culture.

The preferred immunization regimen for effective priming of lymph nodecells is initiated by subcutaneous injection of the antigen, emulsifiedin an adjuvant, in the area drained by the target superficial lymph nodeat 2-3 week intervals. At 3-4 days before the fusion, unadjuvanted virusis again injected into the area. The initial inoculation stimulates thelymphoctyes to produce the anti-BHV-1 antibodies, and the subsequentinjection(s) stimulates further cell multiplication. Immunizing 3-4 daysbefore fusion maximizes the number of lymphocytes in metaphase.Determination of the optimum dose of antigen would be within the skillof a person in the art, though 5 ml of 10⁵ pfu per injection has provento be effective.

On the day of fusion, the lymphocytes are harvested and cosuspended in asuitable growth medium with the cells from the immortal cell line,hereafter referred to as the "myeloma cells." The preferred lymphnode:myeloma cell ratio is in the range of about 1:1 to 1:2. Standardpolyethylene glycol (PEG) fusion procedures such as those reported byVan Deusen et al. [Am. Assoc. Vet. Lab. Diagnost., 24th Annual Proc.211-228 (1981), herein incorporated by reference] or modificationsthereof may be employed.

The primary cells recovered from the fusion process are plated in aselective medium containing a screening agent against unfused myelomacells and myeloma x myeloma fusions. Conventional media for this purposeare HMT (hypoxanthine, methotrexate, and thymidine) or HAT(hypoxanthine, aminopterin, and thymidine) in which the methotrexate oraminopterin functions as the screening agent in that it kills thesusceptible myeloma cells. The unfused lymphocyte cells and bovine xbovine fusions will cease to proliferate after about 1 or 2 week's time.In a normal workup, the cells in the selective medium are cultured atabout 37° C. in a CO² -enriched atmosphere at high humidity. Medium isperiodically screened for antibody production and replenished asnecessary. Applicable screening tests for hybridoma selection includeIFA, RIA, immunoelectrophoresis, enzyme-linked immunosorbent assay(ELISA), and indirect ELISA. The selected primary (1°) hybrids may beserially passaged into larger wells or into flasks as cell numbersincrease.

If the hybrids appear not to be stable in terms of antibody production,they are screened for selectability in a subsequent fusion stage to beperformed. This is accomplished by cultivating the hybrids until asuitable number of cells revert to a condition of being sensitive to oneof the screening agents referred to above. The screening agent-sensitivecells are then selected with a second agent which destroys the cellsresistant to the first screening agent. For example, if the primarycells from the first fusion were selected for methotrexate resistance,they could be cultivated until a pretermined population reverted tomethotrexate sensitivity. The methotrexate-resistant cells areconveniently eliminated by cultivation in the presence of 6-thioguanine,thereby leaving the methotrexate-sensitive cells available for the nextstage of fusion and primary cell selection.

The nonsecreting bovine x myeloma cells carried forward from the firstfusion stage described above are subjected to a "re-fusion" with fresh,primed lymphocytes as previously discussed. The fusion products arecultivated in a selection medium, and the bovine x bovine x murine(bovine² x murine) hybrids are periodically assayed for antibodyproduction in the same manner as the bovine x murine hybrids. Thescreening for subsequent selectability and the re-fusion processes arerepeated as necessary until a stable heterohybidoma is identified. Therepeated "back-crossing" serves to increase the degree of bovineness ofthe fusion product which is ultimately recovered. Based upon theobservations of Tucker et al., supra, this phenomenon is believed to bedue to an increased number of bovine chromosomes introduced by virtue ofeach re-fusion.

The 1° hybrids from the final stage of re-fusion are cloned to insurethat all progeny secrete the desired antibody. Cloning is accomplishedby plating or otherwise propagating individual cells selected from thechosen hybridomas. It is conventional for the cloned cultures to besequentially recloned two or more times to insure homogeneity of thecell line and for expansion of the culture. For antibody production, thecloned cells are cultured in vitro in a suitable growth medium, and theantibody is recovered from the supernatant fluid.

The following example are intended only to further illustrate theinvention and is not intended to limit the scope of the invention whichis defined by the claims.

EXAMPLE 1 Immunizations

A subadult Holstein breed bovine was subcutaneously injected with 5 mlof live, low passage BHV-1 10⁵ pfu/ml emulsified with equal volumes ofIncomplete Freunds Adjuvant in the area drained by the targetsuperficial lymph node at 2-3 week intervals. At 3-4 days before fusion,unadjuvanted virus was injected in the area. The right prescapular, leftprescapular, and right prefemoral superficial lymph nodes weresequentially targeted for the first, second, and third fusions,surgically removed under general anesthesia.

Fusions and Cell Culture

Lymph nodes were sectioned and passed through an 80-mesh sieve (cellsorter). The extracted cells were washed and then frozen or immediatelyused for fusion. Fusions were performed by a modification of a standardPEG protocol [Van Deusen et al., supra] using myeloma cell:lymph nodecell ratios of approximately 1, seeded into 96 well plates. All platecultures were fed with 1:1 Dulbecco's minimal essential medium (DMEM)and high glucose with 10% horse serum to conditioned media from fusionpartner cultures appropriate to each fusion. All useful primary cultureswere cloned and subcloned by limiting dilution using an automaticmicropipettor and were controlled-rate frozen in liquid nitrogen. Fusionpartner selection for methotrexate sensitivity was achieved by passagein media containing 6-thioguanine and 8-azaguanine.

The first fusion of SP2/0 murine myeloma cells with the rightprescapular calf lymph nodes cells in a 3:1 mixture, respectively,resulted in two transiently bovine Ig secreting primary cell lines(αBL5-1.Y1B1 and αBL5-1.Y3E6). After their secretion ceased, the lineswere selected for methotrexate sensitivity by passage in mediacontaining 6-thioguanine and 8-azaguanine. The twomethotrexate-sensitive bovine:murine heterohybridomas were combined in a1:1 ratio and mixed with lymph node cells from the immunized andextracted left prescapular lymph node from the same calf used previouslyin a 1:1 ratio. This fusion again resulted in two transiently bovine Igsecreting cell lines (αBL5A1.Y2C2, and αBL5A1.Y5A11). After thesecretion ceased, the lines were selected for methotrexate sensitivityas before. Cells from the two methotrexate-sensitive bovine² x murineheterohybridomas were combined in a 1:1 ratio and fused with fresh lymphnode cells from an immunized, right prefemoral lymph node from the samecalf as before, this time from a 1:2 mixture, respectively. The fusionresulted in 160 bovine³ x murine primary heterohybridomas secretingbovine antibodies reacting strongly in indirect fluorescent antibodytests using BHV-1. Sixty-eight of these heterohybridomas were stable andrecoverable. Three bovine³ x murine heterohybridomas secreted virusneutralizing, bovine IgG1 antibodies as determined byimmunoelectrophoresis. These hybridomas were cloned and subcloned. Thesubclones are maintained as cell lines αBL5C2.870005, αBL5C2.870009, andαBL5C2.870016 at the National Veterinary Services Laboratories in Ames,IA, and have been deposited under the Budapest Treaty in the AmericanType Culture Collection in Rockville, MD, and assigned Accession No.ATCC HB 9907, ATCC HB 9908, and ATCC HB 9909, respectively. Themolecular weights of the heavy and light chains of the antibody producedby 870009 are 49 Kd and 26 Kd, respectively, as determined byprecipitation PAGE (polyacrylamide gel electrophoresis). For theantibody from 870016, the molecular weights are 50 Kd and 25 Kd,respectively. The chain weights of 870005 have not been determined butare expected to be about the same as those of 870009 and 870016.

In parallel with the final fusion sequence described above, freshlymphocytes were also fused with: (1) SP2/0 myeloma cells; and (2) thecombined αBL5-1.Y1B1 and αBLB-1.Y3E6 bovine x murine hybridomas. Theyields of secretial primary hybrids and stable cell lines for theresultant bovine x murine and bovine² x murine fusions were compared tothose for the bovine³ x murine fusions. It is apparent from the resultssummarized in Table I, below, that the success rate for obtaining astable secreter of bovine monoclonal antibody by triple fusion (bovine³x murine) is approximately 10-fold that of either single or doublefusions.

                  TABLE I                                                         ______________________________________                                                      No. of secretial                                                                          No. of                                              Fusion        hybrids     stable hybrids                                      ______________________________________                                        1st (B.sup.1 × M).sup.a                                                               7           7                                                   2nd (B.sup.2 × M).sup.b                                                               4           3                                                   3rd (B.sup.3 × M).sup.c                                                               160         68                                                  ______________________________________                                         .sup.a Conducted in eight 96well plates.                                      .sup.b Conducted in eleven 96well plates.                                     .sup.c Conducted in ten 96well plates.                                   

EXAMPLE 2

At approximately 6 days of age, New Zealand White Rabbits were injectedby the intracardiac route with 1 ml of antibody fluid. Antibody fluidincluded concentrated tissue culture supernatant from each of the threeheterohybridoma subclones obtained in Example 1, similarly treated mediacontrols, anti-BHV-1 bovine antiserum, and fetal bovine serum.Twenty-four hours later, the rabbits were challenged by intracardiacinjection with 1 ml fluid containing approximately 10⁷ pfu of BHV-1.Deaths observed in 3-14 days were considered specific, and protection isdefined as a reduction in mortality of 50%. The results are reported inTable II, below. The differences observed between the percent mortalityfor bovine antiserum and the percent mortality for each of themonoclonal antibodies is presumed to be attributed to the higherneutralization titers of the polyclonal antiserum.

                  TABLE II                                                        ______________________________________                                        Reduction in BHV-1 Specific Rabbit                                            Mortality by Bovine Antibody                                                  Antibody treatment                                                                           Deaths/No. treated                                                                          % Mortality                                      ______________________________________                                        none           45/48         93.8                                             media control  18/22         81.8                                             870005         1/8           12.5                                             870009          4/12         33.3                                             870016          1/14         7.1                                              fetal bovine serum                                                                           10/17         58.8                                             bovine antiserum                                                                              2/39         5.1                                              ______________________________________                                    

We claim:
 1. A stable heterohybridoma comprising a re-fusion product ofa bovine:murine heterohybridoma and a bovine lymphocyte which had beenprimed to secrete antibodies against bovine herpesvirus-1, saidheterohybridoma characterized by the production of neutralizingmonoclonal antibodies to bovine herpesvirus-1.
 2. A stableheterohybridoma as described in claim 1 and having all the identifyingcharacteristics of a cell line selected from the group consisting ofαBL5C2.870005 (ATCC HB9907), αBL5C2.870009 (ATCC HB9908), andαBL5C2.870016 (ATCC HB9909).
 3. A method for producing a stableheterohybridoma characterized by the production of neutralizingmonoclonal antibodies to bovine herpesvirus-1, comprising the steps:a.harvesting lymphocytes from a bovine animal immunized against bovineherpesvirus-1 and fusing said lymphocytes with cells from an immortalcell line, and selecting for monoclonal antibody-producing,bovine:immortal cell line heterohybridomas; b. culturing thebovine:immortal cell line heterohybridoma from step (a) under monoclonalantibody-secreting conditions until detectable antibody secretionceases; c. harvesting lymphocytes from a bovine animal immunized againstbovine herpesvirus-1 and fusing said lymphocytes with cells of theheterohybridoma from step (b), and selecting for re-fusion productsproducing monoclonal antibodies neutralizing bovine herpesvirus-1; d.repeating steps (b) and (c) until a re-fusion product is stable forantibody production; and e. recovering said stable re-fusion product asthe stable heterohybridoma.
 4. A method as described in claim 3 whereinsaid immortal cell line is of murine origin.
 5. The heterohybridomaproduced by the method of claim
 4. 6. A method as described in claim 4wherein said immortal cell line is SP2/0.
 7. The heterohybridomaproduced by the method of claim
 6. 8. A method as described in claim 3wherein the lymphocytes harvested in step (c) are from the sameindividual animal as the lymphocytes harvested in step (a).
 9. Theheterohybridoma produced by the method of claim
 8. 10. Theheterohybridoma produced by the method of claim 3.